Research On Enrichment Of Circulating Tumor Cells By Size Filtration And Affinity Capture Based On Micro/Nano Technology

The isolation and analysis of circulating tumor cells(CTCs)has great value in the early diagnosis,analysis,treatment and prognosis of cancer.The rapid development of micro/nano technology provides a strong technical support for the isolation and analysis of CTCs.However,the existing micro/nano technology needs further development in terms of purity of captured tumor cell,separation and identification of small-sized tumor cells,and processing of largevolume blood samples.In this paper,from the design of the microcavity structure on the filter membrane,the separation and identification of tumor cells mediated by fluorescent microspheres,and the magnetic separation of the multi-layer filter membrane,we study the application of filter devices to separate and analyze CTCs.This study will provide a reliable experimental basis for the application of micro/nano technology in cancer diagnosis and clinical trials.(1)Pyramidal microcavity array can effectively improve the purity of captured tumor cellsAffinity-based CTCs capture has high purity,but its flux is low.And once tumor cells undergo epithelial-mesenchymal transformation,the capture efficiency is greatly reduced.A simple and effective method for separating CTCs from blood samples by using the physical characteristics of cells is filtration.However,the purity of CTCs separated by various filters prepared by researchers at this stage cannot meet the requirements.To this end,we fabricated a microfluidic filtration device integrated with a silicon-based filter,which showed outstanding capture efficiency and superior enrichment purity when employed to separate CTCs from whole blood samples.We fabricated the silicon filter with pyramidal microcavity array by microfabrication.We designed the structure of the cavity to efficiently enrich tumor cells,while allowing hematologic cells to deform and pass through.The capture efficiency of MCF-7,SW620 and Hela cells spiked in 1 m L of whole blood were approximately 80%.Unwanted white blood cells trapped on the microcavity were below 0.003%.In addition,this microfluidic device successfully identified CTCs in 5 of 6 patients' blood samples,with a range of 5-86 CTCs per m L.These results reveal that the disposable microfluidic device can effectively enrich tumor cells with different sizes and various morphologies,while maintaining high capture efficiency and purity.Therefore,this label-free technique can serve as a versatile platform to facilitate CTCs analysis in diverse biochemical applications.(2)Fluorescent microspheres can not only improve the capture efficiency of small-sized tumor cells,but also can be used to identify tumor cellsA size-based filtration method for separating CTCs from blood sample is based on the fact that CTCs are usually larger than blood cells,and cells below a predetermined size threshold can be removed through the filter membrane.However,some CTCs are almost the same size as white blood cells(WBCs)or even smaller,so it is difficult to separate and detect them by size.To this end,on the basis of pyramidal microcavity array we prepared,we designed a fluorescent microsphere-mediated separation and analysis method of CTCs.This approach can not only separate CTCs(especially small-sized CTCs)in the blood of cancer patients.Moreover,the identification of CTCs can be achieved without immunostaining.This approach used antibody-functionalized fluorescent polystyrene microspheres that can selectively bind to CTCs.The binding of CTCs and fluorescent polystyrene(PS)microspheres leaded to the formation of complexes of CTCs and fluorescent PS microspheres,thereby the CTCs were size-amplified and labeled simultaneously.The pyramidal microcavity array filter device can effectively isolate microspheres-labeled CTCs,while allow hematologic cells to deform and pass through.Using this approach,CTCs were isolated and identified in 15 of 18 patients with metastatic colorectal cancer.This approach will open new possibilities for CTCs isolation and identification and can serve a versatile platform to facilitate CTCs analysis in diverse biomedical applications.(3)High-density gradient magnetic separation device continuously separates circulating tumor cells in bloodExisting filtration technologies can only process a small amount of blood(a few milliliters),it may cause a missed test if there is no CTCs in the collected blood.To this end,we prepared a high gradient magnetic separator for continuous separation of CTCs from the blood samples.Based on the filter device we prepared earlier,we designed a multi-layer filter device.The pore size of the nickel-based filter placed in the filtration chamber gradually decreases from top to bottom,and the separation efficiency is improved while avoiding clogging.We also covered the nickel-based filter with functional magnetic beads,which is more conducive to the capture of CTCs.Experiments show that our high gradient magnetic separator can process large volumes of blood and has the potential to continuously separate the CTCs from the blood to cut off the tumor metastasis pathway.In summary,by designing the microcavity structure on the filter membrane,the purity of the isolated tumor cells can be effectively improved.The combination of the functionalized fluorescent microspheres and the filter membrane not only contributed to the separation of small-sized tumor cells,but also enabled the identification of the isolated tumor cells without immunostaining.By using multi-layer filter magnetic separation device,the blocking phenomenon of traditional single-layer filter when processing a large number of sample can be avoided,which is conducive to obtaining a large number of CTCs samples for subsequent nucleic acid analysis and drug sensitivity experiments.It is also expected to be used for the removal of circulating tumor cells in vivo,thus cutting off the potential of tumor metastasis pathway.